Project Summary/Abstract Most forms of heart disease are associated with a breakdown of the cardiac endothelial barrier, resulting in a damaging leakage and buildup of fluid between blood vessels and the heart tissue (also known as cardiac edema). Treatment has largely focused on heart muscle tissue as a therapeutic target but the endothelium, which plays a critical role in the health of the heart, has been neglected. Multiple studies have demonstrated the involvement of Cx43 in endothelial barrier function and permeability. Preliminary data shows that the Carboxyl-terminus (CT) of Cx43 interacts with both tight junction scaffolding protein Zonula Occludens 1(ZO-1) as well as native Cx43 itself. Furthermore, evidence suggests that endothelial cells might generate freely acting small CT fragments as an endogenous mechanism to self-regulate barrier properties. Using both classical and automated in vitro methods of measuring resistance/impedance across cell monolayers, we demonstrated that ?CT1, a Cx43 CT mimetic peptide, prevented VEGF-mediated breakdown of barrier function and stabilized tight junctions in a model of Age-Related Macular Degeneration (AMD). Moreover, utilizing Electric Cell-substrate Impedance Sensing (ECIS), an automated, impedance-based method used to study the activities of cells grown in culture in real time, we determined that ?CT1 recovered barrier function in a human microvascular endothelial cell line (HMEC1). Recent experiments from our lab indicated the presence of hitherto uncharacterized, naturally occurring ?CT1-like Cx43 CT fragments, which might be packaged within small, extracellular vesicles called exosomes. Our overarching goal is to determine how endothelial cells might utilize an endogenously generated ?CT1-like fragment to affect barrier function. Our specific aims are to: 1) Test the hypothesis that Cx43 CT mimetic peptides protect barrier function via interaction with: 1) ZO-1 and/or 2) Cx43 and 2) Test the hypothesis that endothelial cells generate naturally occurring ?CT1-like Cx43 CT fragments. Exogenous application of Cx43 CT mimetic peptides, ?CT1 and variants (that are either ZO-1 and/or Cx43-binding incompetent) will be used as a tool, to instruct us on the homeostatic functions and mechanisms of endogenous Cx43 and its interactions with binding partners. Western blotting, immunoprecipitation, confocal microscopy, exosome isolation, cryo-electron microscopy and tandem mass spectroscopy will be employed to determine the presence or absence of ?CT1-like Cx43 CT fragments in exosomes derived from Cx43-eGFP Hela cell line expressing wildtype Cx43 and a Cx43- expressing HMEC1 cell line, as well as from ex vivo cardiac preparations. Understanding how the Cx43 CT affects barrier function could be key to advancing novel endothelial barrier stabilizing therapeutics for treating cardiac edema. Training for this project will utilize core facilities and resources shared across Virginia Tech Carilion Research Institute in Roanoke, VA and Virginia Tech's main campus in Blacksburg, VA. A number of highly experienced academics at Virginia Tech, and institutions outside of Virginia Tech, are prepared to aid in the execution of this 3-year project by providing training and other key resources.